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Article An Efficient, Counter-Selection-Based Method for Prophage Curing in Pseudomonas aeruginosa Strains

Esther Shmidov 1,2, Itzhak Zander 1,2, Ilana Lebenthal-Loinger 1, Sarit Karako-Lampert 3 , Sivan Shoshani 1,2 and Ehud Banin 1,2,*

1 The Mina and Everard Goodman Faculty of Sciences, Bar-Ilan University, Ramat-Gan 5290002, Israel; [email protected] (E.S.); [email protected] (I.Z.); [email protected] (I.L.-L.); [email protected] (S.S.) 2 The Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Ramat Gan 5290002, Israel 3 Scientific Equipment Center, The Mina & Everard Goodman Faculty of Life Sciences Bar-Ilan University, Ramat Gan 5290002, Israel; [email protected] * Correspondence: [email protected]

Abstract: Prophages are in the lysogenic state, where the viral is inserted within the bacterial . They contribute to strain genetic variability and can influence bacterial phenotypes. Prophages are highly abundant among the strains of the opportunistic pathogen Pseudomonas aeruginosa and were shown to confer specific traits that can promote strain pathogenicity. The main difficulty of studying those regions is the lack of a simple prophage-curing method for P. aeruginosa strains. In this study, we developed a novel, targeted-curing approach for prophages in P. aeruginosa. In the first step, we tagged the prophage for curing with an ampicillin resistance cassette   (ampR) and further used this strain for the sacB counter-selection marker’s temporal insertion into the prophage region. The sucrose counter-selection resulted in different variants when the prophage- Citation: Shmidov, E.; Zander, I.; cured mutant is the sole variant that lost the ampR cassette. Next, we validated the targeted-curing Lebenthal-Loinger, I.; Karako-Lampert, S.; Shoshani, S.; Banin, E. An Efficient, with local PCR amplification and Whole Genome Sequencing. The application of the strategy resulted Counter-Selection-Based Method for in high efficiency both for curing the Pf4 prophage of the laboratory wild-type (WT) strain PAO1 Prophage Curing in Pseudomonas and for PR2 prophage from the clinical, hard to genetically manipulate, 39016 strain. We believe this aeruginosa Strains. Viruses 2021, 13, method can support the research and growing interest in prophage biology in P. aeruginosa as well as 336. https://doi.org/10.3390/ additional Gram-negative . v13020336 Keywords: Pseudomonas aeruginosa; prophages; bacteriophages; curing; counter-selection; lysogen Academic Editor: Dann Turner

Received: 2 February 2021 Accepted: 18 February 2021 1. Introduction Published: 21 February 2021 Pseudomonas aeruginosa is a Gram-negative, rod-shaped bacterium with a single flagel- lum. It is an opportunistic pathogen of , nematodes, insects, , and humans [1]. Publisher’s Note: MDPI stays neutral It can cause a wide range of acute and chronic infections, such as in severe wounds or with regard to jurisdictional claims in published maps and institutional affil- burns, and chronic lung infections in cystic fibrosis (CF) patients [2,3]. This is enhanced iations. by the bacterium’s low susceptibility to various antimicrobial substances, making most of the infections difficult to treat and life threatening [4,5]. The genetic variability among P. aeruginosa isolates is relatively low; different P. aeruginosa strains share a conserved “core genome” and differ in the variable “accessory segments” [6]. The accessory genome in P. aeruginosa consists mainly of genomic islands and (MGEs) such Copyright: © 2021 by the authors. as , transposons, and prophages-like elements (temperate bacteriophages). Licensee MDPI, Basel, Switzerland. Bacteriophages (phages) are viruses that infect bacteria. They are highly abundant, This article is an open access article distributed under the terms and rapidly spreading, and very diverse biological entities. Phage infections divide into pro- conditions of the Creative Commons ductive lytic or lysogenic (temperate), depending on several factors such as host genetics, Attribution (CC BY) license (https:// phage genetics, environmental conditions, and phage concentration [7]. In the lytic life creativecommons.org/licenses/by/ cycle, phage infection is usually followed by intensive viral DNA replication, eventually 4.0/). leading to a high rate of new phage production and bacterial host lysis. In the lysogenic

Viruses 2021, 13, 336. https://doi.org/10.3390/v13020336 https://www.mdpi.com/journal/viruses Viruses 2021, 13, 336 2 of 12

life cycle, the viral genome is integrated into the bacterial genome as a prophage and further replicates throughout bacterial . A temperate phage can be triggered into the lytic cycle (excision event) spontaneously at a low frequency or by an external stressor such as bacterial DNA damage and physical or chemical factors [7]. The integration of a lysogenic phage into the bacterial genome can influence bacterial phenotypes by providing new traits such as antibiotic resistance, metabolic factors, as well as immunity against specific viral re-infections [7,8]. In some pathogenic bacterial species, prophages encode factors and mediate bacterial adaptation in ecological niches [9]. As mobile elements, prophages are an essential tool for horizontal transfer between bacteria and, among other factors, contribute to strain genetic variability. Most P. aeruginosa strains were identified to contain at least one prophage-like element; some are poly-lysogens, harboring several prophages in their genome [10,11]. Lysogenic phages in P. aeruginosa have been shown to confer selective beneficial traits, such as O antigen conversion, biofilm development, and virulence [12–14]. The PAO1 laboratory strain contains Pf1-like filamentous phage (Pf4) as a prophage that plays a role in small colony variant (SCV) formation during P. aeruginosa biofilm development and has a symbiotic role in bacterial biofilm formation [15,16]. Moreover, 39016 is a clinical isolate of P. aeruginosa that phenotypically differs from the laboratory wild-type (WT) strain PAO1; it exhibits a slower growth rate, reduced motility, and higher biofilm production levels [17]. The prophage-region screening of the published genome of the 39016 strain revealed that it contains five different prophage-like regions in its genome, and four of them are predicted to be intact and able to create infectious virions. As 39016 and PAO1 share the high conserved "core genome" and considering the phenotypic variation among these two strains, the prophage regions of 39016 are likely to influence the bacterial physiology and virulence. A comparison of isogenic strains that differ only in their prophage region can con- tribute to understanding the biological role of a specific phage to the observed traits, the overall prophages contribution, and the cross-regulation between the prophages in poly- lysogenic strains. The main difficulty for this type of study is the lack of a simple and efficient experimental method for curing prophages in P. aeruginosa strains. Previously described prophage curing methods, both for Gram-negative and Gram- positive bacteria, mostly relied on promoting the phage loss with the use of DNA-damaging reagents and the activation of the SOS-response and thus potentially causing a variety of genomic mutations in the bacterial genome, which might result in further variation of the phenotypes [18,19]. Here, we introduce a simple, non-SOS based, highly efficient prophage curing method in P. aeruginosa, using the Gram-negative popular counter-selection marker sacB [20]. We applied the prophage curing method on Pf4 from the WT strain PAO1 and a prophage from the clinical isolate 39016, herein termed PR2. The method can theoretically be applied for all the Gram-negative bacteria containing an intact, excisable prophage in their genome, allowing a better investigation of the prophages and their contribution to bacterial physiology and virulence.

2. Materials and Methods 2.1. Bacterial Strains, Plasmids and Growth Media The bacterial strains and plasmids used in this study are listed in Table1. Primers are listed in Supplementary Table S1. All strains were grown on LB (Luria-Bertani broth, Difco, Sparks, MD, USA) at 37 ◦C unless otherwise specified. For the insertion processes, the following media were used: Vogel Bonner Minimal Medium (VBMM) [21], Pseudomonas Isolation Agar (PIA, Difco, Sparks, MD, USA), and No salt Luria-Bertani (NSLB) + 10% sucrose. For the curing assay and DH5α heat shock, BHI (Brain Heart Infusion broth, Difco, Sparks, MD, USA) media was used. Antibiotic concentrations used in this study were 300 µg/mL carbenicillin (Crb) and 50 µg/mL gentamicin (Gm) for P. aeruginosa and 100 µg/mL ampicillin (Amp) and 30 µg/mL Gm for . Viruses 2021, 13, 336 3 of 12

Table 1. Strains and plasmids used in the study.

Strain or plasmid Description Source P. aeruginosa strains PA01 WT [22] PAO1 ∆Pf4 PAO1, ∆Pf4 This study PAO1 ∆pfiT PAO1, ∆PAO729::Crbr [23] LMG 27,647 P. aeruginosa (Schoeter 1872) BCCM-biological origin: 39016 migula 1900 AL keratitis patients 39016/PR2_ampR 39016, ∆PA39016_000100015::Crbr This study 30916 ∆PR2/Gmr 39016, ∆PR2::Gmr This study 30916 ∆PR2 39016, ∆PR2 This study E. coli strains F- Φ80lacZ∆M15 ∆(lacZYA-argF) U169 DH5α recA1 endA1 hsdR17 (rK-, mK+) phoA Bio-Lab supE44 λ- thi-1 gyrA96 relA1. E. coli S17 thi, pro, hsdR, recA::RP4 S17 [24] -2-Tc::Mu aphA::Tn7, λ-pir, Smr, Tpr Plasmids Crbr (for P. aeruginosa), Ampr (for E. coli), pUCP18-Ap [25] overexpression plasmid, lacZ promoter Gmr and Cmr, pEX18Gm containing a pDONRPEX18Gm HindIII flanked, attP cloning site from [21] pDONR201 Cmr and Gmr, PA39016_000100015 upstream and downstream fragments pDONER/AmpRin_PR2 with AmpR cassette inserted into This study pDONRPEX18Gm by Gateway recombination Cmr and Gmr, PA39016_000100043 and PA39016_000100035 fragments inserted pDONER/PR2_SacB This study into pDONRPEX18Gm by Gateway recombination Cmr and Gmr, PA0725 upstream and downstream fragments inserted into pDONER/Pf4_SacB This study pDONRPEX18Gm by Gateway recombination r for resistance.

2.2. DNA Manipulation and Plasmid Construction Genomic extraction was performed using the DNeasy Blood & Cell Culture DNA Kit (Qiagen, Hilden, Germany). For DNA fragment amplification, Phusion High-Fidelity DNA Polymerase (Thermo) was used. DNA fragments upstream and downstream to the deletion/insertion region were amplified from genomic DNA using PCR. The amplified inserts were cleaned using NucleoSpin Gel and PCR Clean-up (MACHEREY-NAGEL). ampR cassette was amplified from the plasmid pUCP18-Ap using PCR (primers ampR_F and ampR_R). The segments were inserted into pEX18Gm-GW plasmid by recombination (Gateway recombination system and BP Clonase enzyme, Invitrogen, Carlsbad, CA, USA). The DNA Polymerase ReddyMix PCR Kit and M13_F and M13_R universal primers were used to verify successful plasmid transformations. The plasmids were extracted with QIAprep spin miniprep kit (QIAGEN, Hilden, Germany) and sequenced. All of these processes were carried out according to the manufacturer’s instructions.

2.3. Insertion of ampR into Phage Region The PAO1/Pf4_ampR (∆pfiT) and 39016/PR2_ampR strains construction was carried out as previously described [21] with minor changes using the ampR cassette. In PR2 Viruses 2021, 13, 336 4 of 12

prophage, the ampR cassette was inserted instead of the PA39016_000100015 gene. For Pf4 phage curing, the ampR gene was inserted inside the Pf4 locus at ORF PA0729 [23].

2.4. Prophage Curing and Selection for Phage-Cured Mutants The ampR-containing strains were further inserted with sacB into the phage region, as previously described with some process adaptation changes [21]. Briefly, merodiploids with the pDONER/Pf4_SacB or the pDONER/PR2_SacB plasmid undergo site-specific integration into the Pf4 and PR2, respectively, by vector-encoded homologous sequences of prophage region. The merodiploids were streaked on NSLB plates containing 10% sucrose and were incubated for 24 h at 30 ◦C. Single colonies isolated on the sucrose agar were taken and transferred to PIA, LB Gm, and LB Crb plates and incubated overnight. Positive P. aeruginosa colonies that contained the desired deletion grew on PIA plates, but neither on LB Gm nor on LB Crb containing plates. The mutants were verified for complete loss of the phage by PCR for prophage and the intact bacterial attachment site attB.

2.5. Plasmid Curing For recombinant plasmid curing, the 39016 ∆PR2/Gmr culture was incubated overnight in BHI at 42 ◦C with shaking and then streaked overnight on LB plate. Single colonies isolated on LB plates were then transferred to the LB and LB Gm plates for Gm-sensitive variant identification.

2.6. Phage Extraction LB (2 mL) was inoculated with bacterial strain and incubated overnight. The next day, the bacteria were diluted 1:50 with medium to a final volume of 4 mL and incubated for 3 h. Then, 1 mL of bacteria was centrifuged at 14,000× g for 2 min, and 900 µL of the supernatant was filtered using a 0.45 µm filter (Whatman, Maidstone, Kent, UK).

2.7. Plaque Assay LB (2 mL) was inoculated with recipient strain and incubated overnight with shaking. The next day, bacteria were diluted 1:50 with medium to a final volume of 2 mL and incubated with shaking for 2 h. Further, 100 µL of the bacteria was transferred into 5 mL heated (50 ◦C) LB with 0.5% agar, and then gently mixed and poured onto the surface of a 1.5% LB-agar plate. Serial dilutions (1:10) of the extracted phage stock were made, droplets of 2 µL were spotted on to the top-agar layer. The plate was incubated overnight until plaques were formed.

2.8. Whole-Genome Sequencing DNA was extracted from cell pellets using the DNeasy Blood & Tissue Kit (Qiagen), and DNA quality was evaluated by gel electrophoresis. The library was constructed with NEBNext Ultra II FS DNA Library Prep Kit for Illumina (NEB, UK) according to the manufacturer’s instructions using 500 ng as the starting material. DNA was fragmented 20 min with three cycles of PCR. The final quality was evaluated by TapeStation High Sensitivity D1000 Assay (Agilent Technologies, CA, USA). Sequencing was performed based on Qubit values and loaded onto an Illumina MiSeq using the MiSeq Micro 150× 2 kit (Illumina, CA, USA).

2.9. Genome Assembly and Sequence Analysis The MiSeq sequencing resulted in 1.13–2.87 million paired-end reads per strain. FastQC (v0.11.2) (https://www.bioinformatics.babraham.ac.uk/projects/fastqc(accessed on 11 February 2021)) was used to assess the quality of the raw reads. For each strain de novo assembly was performed by SPAdes (v3.13.0) [26] with parameters -k 21,33,55,77,99, 127 –careful. QUAST (v5.0.2) [27] was used for quality assessment of the genome assem- blies. Contigs with a minimal length of 500 bases from the assemblies of P. aeruginosa 39016 (WT and mutant) and P. aeruginosa PAO1 (WT and mutant) were reordered according to reference (NCBI Reference Sequence NZ_CM001020.1 and NC_002516.2, respec- Viruses 2021, 13, x FOR PEER REVIEW 5 of 13 Viruses 2021 , 13, 336 5 of 12

was used for quality assessment of the genome assemblies. Contigs with a minimal length of 500 bases from the assemblies of P. aeruginosa 39016 (WT and mutant) and P. aeruginosa tively) using Mauve aligner [28,29]. Regions of prophage insertion were determined using PAO1 (WT and mutant) were reordered according to reference genomes (NCBI Reference PHASTER [30]. BLAST Ring Image Generator (BRIG) (https://sourceforge.net/projects/ Sequence NZ_CM001020.1 and NC_002516.2, respectively) using Mauve aligner [28,29]. brig/Regions (accessed of prophage on 11 Februaryinsertion were 2021)) determined [31], and using Mauve PHASTER [28] were [30]. used BLAST to compareRing Image the WT andGenerator mutant to(BRIG) the reference (https://sourceforge.net/proje genome. cts/brig/) [31], and Mauve [28] were used to compare the WT and mutant to the reference genome. 3. Results 3.1.3. The Results Targeted Curing Principle 3.1.The The overall Targeted principle Curing Principle of the curing strategy is presented in Figure1. The first step was to tagThe theoverall targeted principle prophage of the curing with strategy an ampR is presentedcassette in insertion Figure 1.into The first the step phage was region usingto tag an the allelic targeted replacement prophage with technique. an ampR After cassette creating insertion the into ampicillin-resistant the phage region using strain, it wasan further allelic replacement used for sacB technique.insertion After by creating utilizing the theampicillin-resistant site-specific integration strain, it was step fur- of the gentamicinther used resistancefor sacB insertion marker by (utilizingaacC1) and the site-specificsacB containing integration plasmid step backbone of the gentamicin in theallelic replacementresistance marker method. (aacC1 Homologous) and sacB containing regions wereplasmid used backbone to direct in the the allelic backbone replacement integration intomethod. the prophage, Homologous and regions in this were way, used a temperate to direct ampR/sacB/aacC1the backbone integrationcontaining-form into the pro- of the prophagephage, and was in created this way, (see a temperate scheme in ampR/sacB/aacC1 Figure1). To pushcontaining-form toward either of the insertion prophage or the was created (see scheme in Figure 1). To push toward either insertion or the desired pro- desired prophage curing, the merodiploids were streaked on sucrose plates. The sucrose phage curing, the merodiploids were streaked on sucrose plates. The sucrose counter-se- ampR counter-selectionlection resulted in resulted either no in change either noin the change ampR in-inserted the prophage-inserted (Figure prophage 1A), integra-(Figure1A) , integration-sitetion-site deletion deletion mutants mutants (Figure 1B), (Figure sacB1 mutantsB), sacB thatmutants survived that the survivedsucrose selection the sucrose selection(Figure (Figure1C), or the1C), whole or the prophage-cured whole prophage-cured variant (Figure variant 1D). Out (Figure of all 1theseD). Outoptions, of all the these options,prophage-cured the prophage-cured mutant is the mutant sole Crb-sensitive is the sole variant Crb-sensitive (Figure 1). variant Such a system (Figure makes1). Such a systemit possible makes to itselect possible for rare to colonies select for that rare lost colonies the ampR that-inserted lost prophage. the ampR -insertedThe separation prophage. Theof separationthe two steps of was the to two assure steps insertion was to distance assure between insertion the distance counter-selection between and the the counter- selectionpositive-selection and the positive-selection markers to avoid false-positive markers to avoid results false-positive in variants that results lost all inthe variants mark- that losters all but the not markers the whole but prophage. not the whole prophage.

Figure 1. The targeted curing methodFigure 1. principle. The targeted The curing prophage method is pr borderedinciple. The by prophage the right is attachment bordered by site the attRright(dark attachment green) and the left attachment site attLsite(orange). attR (dark The green) first stepand the is ampR left attachment(red) insertion site attL for (orange). prophage The tagging. first step The is ampR second (red) step inser- is the tion for prophage tagging. The second step is the integration of the plasmid backbone, containing integration of the plasmid backbone,accC1 (gray) containing and sacBaccC1 (light(gray) blue) and intosacB the prophage(light blue) region into via the homologous prophage region recombination. via homologous Next, recombination. Next, the temporal tagged prophage with the integrated sacB undergoes counter-selection by growing on the sucrose-containing growth medium. The counter-selection outcome can be either (A) unchanged tagged prophage, (B ) integration site deletion (marked with an asterisk), (C) sacB mutated variant (asterisk), or (D) prophage curing. Notably, only option (D) would be Crb sensitive. Viruses 2021, 13, 336 6 of 12

3.2. Pf4 Phage of PAO1 Curing The curing of Pf4 prophage of the PAO1 strain was carried out to demonstrate the efficiency of the targeted curing method. First, PAO1/Pf4_ampR (∆pfiT) mutant was created by inserting the ampR cassette instead of the PAO729 gene, which encodes for the PfiT toxin protein from the toxin– antitoxin (TA) pfiT/pfiA system. The insertion did not alter the bacterial growth compared to WT PAO1, and the strain still showed spontaneous phage loss [23]. To further integrate the sacB-containing plasmid backbone, the upstream and down- stream homologous sequences of the PA0725 gene were used to direct the insertion into the prophage and create the transient ampR/sacB/accC1 containing Pf4-containing prophage. The counter-selection on sucrose-containing medium resulted in a high yield; 100% of the analyzed colonies (40/40) were both Crb- and Gm-sensitive, indicating a potential phage loss. Complete loss of the Pf4 prophage and the circular replicative form (RF) DNA was confirmed for this mutant by PCR amplification around the attB site of Pf4, the attR site for the integrated form, and the phage attachment site (attP) for the RF form of Pf4 phage (Figure2A–C) . One such mutant was randomly chosen and designated as ∆Pf4 strain. As the WT PAO1 strain exhibits immunity properties against Pf4 re-infection, the ∆Pf4 strain was tested for immunity loss. The cured strain infection with Pf4 phages, extracted from WT PAO1, resulted in plaque Viruses 2021, 13, x FOR PEER REVIEW 7 of 13 formation and bacterial susceptibility, unlike the WT strain (Figure2D).

Figure 2. Pf4 curing verification. (A-C) Curing verification by PCR, Lanes 1–2 represent the DNA Figure 2. Pf4 curing verification. (A-C) Curing verification by PCR, Lanes 1–2 represent the DNA of of randomly picked Crb-sensitive colonies, and lane 3 represents the WT PAO1 for control. (A) randomlyAmplification picked of 1800 Crb-sensitive bp around colonies,the Pf4 attB and site. lane (B) 3Amplification represents the of 1000 WT PAO1bp around for control.the Pf4 attR (A) Amplifi- cationsite that of can 1800 only bp be around amplified the in Pf4 theattB integratedsite. (B )form. Amplification (C) Amplification of 1000 of bp 750 around bp around the the Pf4 Pf4attR site that canattP only site that be amplified can only be in amplified the integrated in the form.RF form. (C )(D Amplification) Curing verification of 750 by bp plaque around assay, the Pf4 serialattP site that dilutions of Pf4 phage are extracted from WT PAO1 strain used to infect WT PAO1 and ∆Pf4 can only be amplified in the RF form. (D) Curing verification by plaque assay, serial dilutions of Pf4 strain. phage are extracted from WT PAO1 strain used to infect WT PAO1 and ∆Pf4 strain. To assure the genome integrity of this strain and to prove once more the phage loss, whole-genome sequencing was performed to the cured strain, and the resulting assembly was aligned to the PAO1 WT sequence. The genome analysis revealed that the WT strain assembly covers 99.284% of the reference genome and that besides the Pf4 region, no ad- ditional significant insertions or deletions were detected in the mutant strain (Figure 3).

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To assure the genome integrity of this strain and to prove once more the phage loss, whole-genome sequencing was performed to the cured strain, and the resulting assembly was aligned to the PAO1 WT sequence. The genome analysis revealed that the WT strain assembly covers 99.284% of the reference genome and that besides the Pf4 region, no additional significant insertions or deletions were detected in the mutant strain (Figure3).

Figure 3. The ∆Pf4 strain differs from WT in the Pf4 region. Genomic alignment with PAO1 strain is used as a reference, PAO1 WT sequence assembly is indicated in green, and ∆Pf4 strain sequence assembly is indicated in blue. Prophage regions are labeled in red; coordinates are taken from Figure 3. The ∆Pf4 strain differs from WT in the Pf4 region. Genomic alignment with PAO1 strain PHASTER analysis to the reference PAO1 strain. The cured region is marked with an arrow. We note is used as a reference, PAO1 WT sequence assembly is indicated in green, and ∆Pf4 strain se- that there is a difference between our laboratory WT strain and the reference strain around region quence assembly is indicated in blue. Prophage regions are labeled in red; coordinates are taken 2800from bp, but PHASTER this difference analysis is to identical the reference in the Pf4-mutantPAO1 strain. strain. The cured region is marked with an arrow. 3.3.We PR2 note Phage that of there 39016 is a Curing difference between our laboratory WT strain and the reference strain around region 2800 bp, but this difference is identical in the Pf4-mutant strain. Clinical isolates of P. aeruginosa are less attenuated than the WT PAO1 strain; thus, the genetic3.3. manipulationsPR2 Phage of 39016 are limited Curing and challenging in those isolates. In order to demonstrate the efficiency of our method for a clinical isolate, the 39016 strain of P. aeruginosa was tested Clinical isolates of P. aeruginosa are less attenuated than the WT PAO1 strain; thus, for curing its second prophage (PR2). the genetic manipulations are limited and challenging in those isolates. In order to demon- First, 39016/PR2_ampR mutant was created by inserting the ampR cassette instead of thestrate PA39016_000100015 the efficiency of our gene meth (hypotheticalod for a clinical protein, isolate, herein the termed 39016 as strain AmpRin of P. region). aeruginosa Next,was to tested assure for that curing the insertion its second did prophage not affect (PR2). the excision ability of PR2, a comparison of the infectivityFirst, 39016/PR2_ of phages producedampR mutant by WT was 39016 created or byby 39016/PR2_inserting theampR ampRstrain cassette has instead been of performed.the PA39016_000100015 The results showed gene that (hypothetical the cassette’s protei insertionn, herein did termed not impair as AmpRin the bacterial region). growthNext, or to the assure infectious that the phage insertion production did not of theaffect 39016 the strainexcision (Figure ability S1). of PR2, a comparison ofFurther the infectivitysacB integration of phages was produced directed by by WT homologous 39016 or by sequences 39016/PR2_ upstreamampR strain and down- has been streamperformed. to the intragenicThe results region showed of that PR2 the located cassette's between insertion the PA39016_000100034did not impair the bacterial and PA39016_000100035growth or the infectious genes (SacBin phage production region). After of the the 39016 sucrose strain counter-selection, (Figure S1). the an- tibiotic screenFurther revealed sacB integration approximately was 70%directed (57/80) by Crb-sensitivehomologous coloniessequences that upstream were also and Gmdownstream resistant. Prophage to the intragenic gene amplification region of PR2 indicated located that between the phage the PA39016_000100034 was lost (Figure4A); and however,PA39016_000100035 the SacBin region genes was (SacBin still present region). in the After cured the strainsucrose (Figure counter-selection, S2) indicating the some antibi- otic screen revealed approximately 70% (57/80) Crb-sensitive colonies that were also Gm

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resistant. Prophage gene amplification indicated that the phage was lost (Figure 4A); how- ever, the SacBin region was still present in the cured strain (Figure S2) indicating some recombinant plasmid has been created in the process that contains the accC1 marker and recombinant plasmid has been created in the process that contains the accC1 marker and the SacBin region; this strain was named “∆PR2/GmR.” In order to cure the recombinant the SacBin region; this strain was named “∆PR2/GmR.” In order to cure the recombinant plasmid, we applied a temperature-based plasmid curing assay on ∆PR2/GmR strain. PCR plasmid, we applied a temperature-based plasmid curing assay on ∆PR2/GmR strain. PCR confirmed the resulting Gm-sensitive colonies for the SacBin region loss and attB region confirmed the resulting Gm-sensitive colonies for the SacBin region loss and attB region amplification for the complete phage loss (Figure4B). One such mutant was chosen for amplificationstudy and designated for the complete as ∆PR2 phage strain. loss (Figure 4B). One such mutant was chosen for study and designated as ∆PR2 strain.

Figure 4. PR2 curing verification by PCR. (A) AmpRin region amplification results either in null, Figure 4. PR2 curing verification by PCR. (A) AmpRin region amplification results either in null, 2000 bp, or 1200 bp in the 2000 bp, or 1200 bp in the cured, ampR-containing, or WT strain, respectively; lanes 1–8 represent cured, ampR-containing, or WT strain, respectively; lanes 1–8 represent randomly picked Crb-sensitive colonies, lanes 9–10 randomly picked Crb-sensitive colonies, lanes 9–10 represent Crb-resistant colonies, and the WT represent Crb-resistant colonies,39016 and is for the positive WT 39016 control. is for ( positiveB) Amplification control. ( Bof) Amplification2000 bp around of the 2000 PR2 bp attB around site theof ∆ PR2PR2attB and WT site of ∆PR2 and WT 39016,39016, and positive and positive control control (ctrl) amplification(ctrl) amplification of 324 of bp 324 PA39016_100004 bp PA39016_100004 external external gene. gene.

ToTo verify verify that that both the prophage-curing and and the plasmid-curing processes processes did did not not result in a high mutation rate in the ∆PR2 strain, the cured strain genome was sequenced result in a high mutation rate in the ∆PR2 strain, the cured strain genome was sequenced and aligned to the WT 39016 sequence. The genome analysis revealed that PR2 was missing and aligned to the WT 39016 sequence. The genome analysis revealed that PR2 was miss- in the cured strain, and no additional significant insertions or deletions were detected ing in the cured strain, and no additional significant insertions or deletions were detected (Figure5). Moreover, PHASTER [ 30] analysis of the assembled genome of ∆PR2 revealed (Figure 5). Moreover, PHASTER [30] analysis of the assembled genome of ∆PR2 revealed that the cured strain differs from the WT 39016 in its prophage content solely in the second that the cured strain differs from the WT 39016 in its prophage content solely in the second phage; the curing process did not affect all the other phages of 39016. phage; the curing process did not affect all the other phages of 39016. Unfortunately, the attempts to use the cured ∆PR2 strain as a host by infecting with phages extracted from WT 39016 strain had failed, suggesting some cross-immunity be- tween PR2 and the other prophages in 39016. However, as indicated by PCR amplification of the extracted intact phages, while successful PR2 virion production was observed in the WT strain, it is not produced by the cured strain (Figure S3).

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Figure 5. The ∆PR2 strain differs from WT in the PR2 region solely. Genomic alignment with 39016 strain is used as a Figure 5. The ∆PR2 strain differs from WT in the PR2 region solely. Genomic alignment with 39016 reference, 39016 WT sequence assembly is indicated in green, and ∆PR2 strain sequence assembly is indicated in blue. strain is used as a reference, 39016 WT sequence assembly is indicated in green, and ∆PR2 strain Prophage regions are labeledsequence in red; assembly coordinates is indicated are taken in blue. from Prophage PHASTER regions analysis are to labeled the reference in red; 39016 coordinates strain. Theare curedtaken region is marked with anfrom arrow. PHASTER analysis to the reference 39016 strain. The cured region is marked with an arrow.

4.Unfortunately, Discussion the attempts to use the cured ∆PR2 strain as a host by infecting with phages extracted from WT 39016 strain had failed, suggesting some cross-immunity be- In this study, we provide a simple counter-selection-based curing methodology. The tween PR2 and the other prophages in 39016. However, as indicated by PCR amplification approach was proven successful for both the laboratory PAO1 WT strain and the clinical, of the extracted intact phages, while successful PR2 virion production was observed in the hard to genetically manipulate, 39016 strain. The guiding principles of the targeted method WT strain, it is not produced by the cured strain (Figure S3). were first to avoid DNA damaging as in the usage of SOS response promoting agents, and second, to cure a specific prophage without influencing the other prophages in the genome 4. Discussion of the poly-lysogenic strains. In thisThe study, strategy we provide for prophage a simple targeted counter-selection-based curing from poly-lysogenic curing methodology. strains should The take approachinto the was similarity proven ofsuccessful the different for both prophages. the laboratory For poly-lysogenic PAO1 WT strain strains and that the carry clinical, different hardprophages, to genetically there manipulate, is no need for39016 a spatial strain. approach; The guiding as we principles have shown of the with targeted 39016, the methodother were prophages first to areavoid not affectedDNA damaging by the curing as in process.the usage For of strains SOS response that carry promoting highly similar agents,prophages, and second, the tagging to cure step a specific should prop be directedhage without to a unique influencing region orthe a low-similarityother prophages region in thewithin genome the of prophage. the poly-lysogenic PAO1 is an strains. example of such a ploy-lysogenic strain as it carries two highlyThe strategy similar for prophages prophage in targeted its genome; curing Pf4 from and Pf6poly-lysogenic prophages [strains32]. We should achieved take 100% into curingthe similarity by focusing of the on different the unique prophages. region of For Pf4 poly-lysogenic for the ampR insertion strains that step carry to assure differ- proper ent prophages,prophage-tagging, there is no and need the for second a spatial step approach; was directed as we into have a commonshown with region 39016, with the high othersimilarity prophages for are both not Pf6 affected and Pf4. by For the strains curing that process. carry For multiple strains copies that carry of the highly same prophage,simi- lar prophages,it should be the recommended tagging step should to sequence be directed properly to a following unique region the tagging or a low-similarity step to identify which of the prophages was eventually targeted to be cured in the second step.

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The use of a counter-selection marker for prophage curing has been described before for pyogenes [33]. The method is based on first creating a streptomycin- resistant strain that is mutated in its rpsL gene, and further insertion of other antibiotic resistance cassette and the rpsL WT allele for counter-selection by streptomycin. The difficulty of using such an approach in P. aeruginosa is mainly because it does not harbor streptomycin resistance through genomic mutations in the rpsL gene [34], so the counter- selection marker is not suitable in this case. Furthermore, the resistant mutant creation process by genomic mutation might disrupt the genomic integrity and cause additional, uncontrolled mutations. Another paper described a different counter-selection-based curing technique in Vibrio natriegens by inserting an inducible ccdB toxin into the phage region and further counter-selection by toxin induction [34]. The usage of ccdB toxin or other toxins from TA modules might be problematic for targeted curing based on the assumption that the toxins might induce other prophages in the genome regardless of the curing process [23]. Our approach avoids such problems. It is important to note that the curing process of PR2 from 39016 had, in addition to the targeted curing process, an additional plasmid curing step to remove the recombinant plasmid produced in the curing process. The creation of the recombinant plasmid might have occurred due to the conservation of an oriT, the atcc1 cassette, and some specific, important regions of the prophage. A similar case was also reported in the curing of specific prophage in S. pyogenes. The produced recombinant plasmid in the reported case was related to the prophages’ cross-regulatory properties. When attempting to cure the same phage, in the background of other cured prophages, the recombinant plasmid was not produced [33]. The curing process of Pf4 from PAO1 was very efficient, with a high yield of cured mutants that can serve as a surrogate for infection from PAO1-produced phages. The high yield might be related to the fact that the first curing step included the deletion of the pfiT toxin gene; in other cases, it might be less efficient due to the addiction mediated by the stable toxin that will promote killing in the cured, antitoxin free variants. Thus, as part of the curing assay, it is recommended to initially scan for TA systems in the specific prophage sequence and target the toxin in the ampR insertion step. Using the double-stage approach, we managed to overcome the difficulty with the limited counter-selection available markers for P. aeruginosa, when in the first stage, the sacB marker was used for plasmid-backbone removal, and then, the same marker was utilized to select for complete phage loss from the curing step. It is important to note that the method can potentially be applied to other mobile genetic elements and stable-plasmids that are commonly associated with bacterial virulence [35,36]. The principle of the method and the high observed efficiency gives a better under- standing of prophage–host interactions. The prophage-excision event mostly attributed to the phage disadvantage for staying integrated [37] or regulated by the host for a temporary extra-chromosomal state (active lysogeny) [38], but here, we showed that under certain conditions, when the prophage’s contents endanger the host, it would prefer to lose the whole prophage rather than the specific risk-containing region. This fact emphasizes the need for curing rather than deletion methods for such regions.

Supplementary Materials: The following are available online at https://www.mdpi.com/1999-4 915/13/2/336/s1, Supplementary methods, Table S1: Primers used in the study, Figure S1: ampR insertion into the PR2 prophage did not alter the bacterial growth and PAO1-infectious phages production, Figure S2: The SacBin region presents in PR2-cured colonies. Figure S3: PR2 phages are not produced in the cured strain. Author Contributions: Conceptualization, E.S., I.Z., and E.B.; methodology, E.S, I.Z., and S.K.-L. data analysis, E.S. and I.L.-L.; writing—original draft preparation, E.S.; writing—review and editing, E.S., S.S., and E.B.; supervision, E.B.; funding acquisition, E.B. All authors have read and agreed to the published version of the manuscript. Viruses 2021, 13, 336 11 of 12

Funding: This work is part of the Ph.D. thesis of E.S. Partial funding for this work was through the Dyna and Fala Weinstock Foundation to E.B., the President’s Scholarships, and the Merit-Based Scholarships at the Institute of Nanotechnology of Bar-Ilan University for E.S. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: We thank Yigal Brochin for editing the article. Conflicts of Interest: The authors declare no conflict of interest.

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